Early Astrocytic Dysfunction Is Associated with Mistuned Synapses as well as Anxiety and Depressive-Like Behavior in the AppNL-F Mouse Model of Alzheimer’s Disease

Background: Alzheimer’s disease (AD) is the most common neurodegenerative disease. Unfortunately, efficient and affordable treatments are still lacking for this neurodegenerative disorder, it is therefore urgent to identify new pharmacological targets. Astrocytes are playing a crucial role in the tuning of synaptic transmission and several studies have pointed out severe astrocyte reactivity in AD. Reactive astrocytes show altered physiology and function, suggesting they could have a role in the early pathophysiology of AD. Objective: We aimed to characterize early synaptic impairments in the AppNL-F knock-in mouse model of AD, especially to understand the contribution of astrocytes to early brain dysfunctions. Methods: The AppNL-F mouse model carries two disease-causing mutations inserted in the amyloid precursor protein gene. This strain does not start to develop amyloid-β plaques until 9 months of age. Thanks to electrophysiology, we investigated synaptic function, at both neuronal and astrocytic levels, in 6-month-old animals and correlate the synaptic activity with emotional behavior. Results: Electrophysiological recordings in the hippocampus revealed an overall synaptic mistuning at a pre-plaque stage of the pathology, associated to an intact social memory but a stronger depressive-like behavior. Astrocytes displayed a reactive-like morphology and a higher tonic GABA current compared to control mice. Interestingly, we here show that the synaptic impairments in hippocampal slices are partially corrected by a pre-treatment with the monoamine oxidase B blocker deprenyl or the fast-acting antidepressant ketamine (5 mg/kg). Conclusions: We propose that reactive astrocytes can induce synaptic mistuning early in AD, before plaques deposition, and that these changes are associated with emotional symptoms.

were then mounted on non-coated microscope glass coverslips using the fluoromount mounting medium (Invitrogen,.Images were taken on a confocal microscope (Zeiss LSM700) with a 10x objective.

Aβ ELISA
The three Aβ fractions were analyzed using a homogenous sandwich ELISA using the Aβ N-terminal specific antibody 3D6 (murine version of Bapineuzumab, produced in-house) as both capture and detection antibody.96-wells ELISA plates were coated with 1 µg/mL 3D6 and incubated over night at +4 °C.Plates were blocked with 1% (w/v) BSA in PBS 1X for 2 h.

Astrocyte density
Astrocyte density was manually evaluated by an experimenter blinded to experimental condition, using the software Image J. Briefly, GFAP-positive cells were counted in Z-projected images.Two images per animal were obtained using confocal imaging.DAPI was used to determine the centroid and to facilitate the identification of GFAP positive cells.Cells with putative nuclei outside of the field were discarded.The density was calculated as follow:

Splash test
The animals were isolated in a new cage and habituated for at least 20 min.200 µL of 10% sucrose solution was squirted on the mouse's snout and the grooming behavior (latency to first, frequency and total time), used to evaluate self-care, was manually scored for 5 min.Mice were placed back together in their home cage after the experiment.

Sucrose preference test
Each animal was singled housed and habituated to drink water from two identical bottles for 48 h.During the following 72 h, mice could choose between water and a 1% sucrose solution.
Sucrose solution intake was measured by weighing the bottles before and after the last 24 h and expressed as the percentage of the total amount of liquid ingested, normalized to the mouse body weight.Each bottle was replaced and switched from left to right daily.

Rotarod test
Animals were tested on a rotating bar of 5 cm diameter on which mice were placed facing the sense of the rotation.Animals were habituated to the device for 30 s at a constant speed of 5 rotations per minute (rpm) the day before the experiment.This was followed by 3 trials per animal with 15 min between each trial.During the trials, mice were placed on an accelerating rod increasing from 5 to 50 rpm for 5 min.The test is stopped when the mouse fall from the rod.The latency before falling and the maximum rotation speed before falling are recorded.
The average of each score gives the final z-score per animal.X: individual values for the considered parameter; µ: mean of the control group; σ: standard deviation of the control group.In this figure, App NL-F + NaCl mice have been used as the control group.
For the three fractions: Two-way ANOVA with the genotype and the brain region as the main factors.Bonferroni multiple comparison: **p<0.01;***p<0.001statistically significant as shown.C57Bl6: n=6, App NL-F : n=6.Panel A: scale bar 200m.Supplementary Figure 2. Astrocyte morphology analysis (related to Fig. 1).Using Z-stack images of GFAP and DAPI staining were merged in a z-projection and were treated according to the presented workflow (A).Before reconstruction astrocytes were manually separated (B): the first image shows the connection (red arrows) on the deconvoluted image while the second image shows the raw image of the connexin 43 (Cx43) staining, use to discriminate connecting hubs between astrocytes.The third image shows the absence of connections after manual removal.(C) depicts how the syncytium looks like before, and after separation.Once all astrocytes have been separated, the morphology of each cell was 3D reconstructed and individually analyzed.(D) shows the number of astrocytes per image, calculated on obtained z-projections (two images per mouse, three mice per group).Supplementary Figure 3. Complementary electrophysiological measurements (related to Fig. 2, 3, and 7).Patch clamp experiments revealed no difference in frequency (A), amplitude (B) decay time (C), or rise time (D) of sEPSC.No differences were unveiled either in decay time (E) nor rise time (F) of mEPSC.Representative traces for sEPSC are depicted under the graphs.C57Bl6 controls: n=10 cells recorded in seven animals, App NL-F : n=10 cells recorded in seven animals.Pretreatment with deprenyl did not affect the frequency of sEPSC (G) but increased the amplitude (H) Representative traces are depicted under the graphs.Neither the rise time or the decay time for sEPSC (I and J, respectively) and mEPSC (K and L, respectively) were affected by deprenyl.App NL-F + aCSF: n=8 cells recorded in four animals; App NL-F + deprenyl: n=8 cells recorded in four animals.No differences were unveiled in frequency (M), amplitude (N), decay time (O) or rise time (P) of sEPSC after ketamine treatment.The NMDA receptor blocker did not affect the decay time (Q) nor the rise time (R) of mEPSC.Representative traces for sEPSC are depicted under the graphs.App NL-F + NaCl: n=7 cells recorded in five animals, App NL-F + ketamine: n=6 cells recorded in six animals.Supplementary Figure 4. Rotarod, splash test and sucrose preference test (related to Figs. 5

Table 1 .
Calculation of the emotionality z-score (related to Fig.5).For each test, a z-score (z = (X − µ)/σ) is calculated for each parameter of consideration (zSC_param).All the zSC-parameters are then averaged per test leading to a test z-score (zSc_test).The average of each score gives the final z-score per animal.X: individual values for the considered parameter; µ: mean of the control group; σ: standard deviation of the control group.In this figure, C57Bl6 mice have been used as the control group.